Staphylococcus aureus increases platelet reactivity in patients with infective endocarditis

Thromboembolism is frequent in infective endocarditis (IE). However, the optimal antithrombotic regimen in IE is unknown. Staphylococcus aureus (SA) is the leading cause of IE. First studies emphasize increased platelet reactivity by SA. In this pilot study, we hypothesized that platelet reactivity is increased in patients with SA− IE, which could be abrogated by antiplatelet medication. We conducted a prospective, observatory, single-center cohort study in 114 patients with IE, with four cohorts: (1) SA coagulase positive IE without aspirin (ASA) medication, (2) coagulase negative IE without ASA, (3) SA coagulase positive IE with ASA, (4) coagulase negative IE with ASA. Platelet function was measured by Multiplate electrode aggregometry, blood clotting by ROTEM thromboelastometry. Bleeding events were assessed according to TIMI classification. In ASA-naïve patients, aggregation with ADP was increased with coag. pos. IE (coagulase negative: 39.47 ± 4.13 AUC vs. coagulase positive: 59.46 ± 8.19 AUC, p = 0.0219). This was abrogated with ASA medication (coagulase negative: 42.4 ± 4.67 AUC vs. coagulase positive: 45.11 ± 6.063 AUC p = 0.7824). Aspirin did not increase bleeding in SA positive patients. However, in SA negative patients with aspirin, red blood cell transfusions were enhanced. SA coagulase positive IE is associated with increased platelet reactivity. This could be abrogated by aspirin without increased bleeding risk. The results of this pilot study suggest that ASA might be beneficial in SA coagulase positive IE. This needs to be confirmed in clinical trials.

Infective endocarditis (IE) is a severe disease, causing destruction of endocardial structures and resulting in high morbidity and mortality 1,2 . Despite all improvements in medical care and therapy, the overall mortality remains high (up to 18%) due to cardiac failure and thromboembolic complications 2 . In vitro studies suggested that increased platelet activation plays a central role in the pathophysiology of IE 3 . These studies showed that IE inducing bacteria can interact with platelets and coagulation factors 4,5 . This facilitates microbial surface binding and formation of vegetations promoting thromboembolic events 6 . Especially Staphylococcus aureus (SA) and its virulence factor clumping factor A (ClfA) have been shown to trigger platelet adhesion and to directly induce platelet aggregation in vitro 7,8 . Therefore, IE inducing bacteria can be divided in SA coagulase positive and coagulase negative bacteria depending on their ability to induce clotting reactions. ClfA allows SA to cover its surface with proteins and platelets from the host organism and to escape immune response 9 . As underlying mechanisms for the pro-thrombotic effects, fibrinogen and IgG dependent pathways were suggested 8,10,11 . Furthermore, SA also influences the coagulation system: it secretes soluble virulence factors, like staphylocoagulase (Coa) and von Willebrand factor-binding protein (vWbp) that directly interact with prothrombin, leading to the conversion of fibrinogen to fibrin 10,12,13 . However, investigation on platelet function in IE-patients is rare. Therefore, we compared platelet function of patients with SA coagulase positive (SA+) and coagulase negative (SA−) IE. Furthermore, we investigate the effects of acetylsalicylic acid (ASA) as antiplatelet medication in these patients.

Methods
Study design, patient population. In this observatory, single-center cohort study, we analysed 114 patients with clinically proven IE with indication for valve surgery in the University Hospital Düsseldorf, Germany from 2013 to 2017. Informed consent was obtained from all patients. Germs were detected in blood cultures or by microbiological examination of the explanted valve respectively. Four study groups were defined depending on prior and perioperatively continued ASA medication (oral administration of 100 mg ASA once per day) and SA status (Fig. 1). Age under 18, coagulation disorders, haematological diseases and platelet medication others than ASA were exclusion criteria. After surgery, patients were on oral anticoagulation for 3 months, followed by indefinite single antiplatelet medication. The study conformed to the declaration of Helsinki and was approved by the Ethics Committee of the University of Düsseldorf (2018-105-RetroDEuA).
Platelet function and coagulation parameters testing. Blood samples were collected in citrate vacutainers (1:10) immediately before valve surgery. Platelet function was measured as point of care test by multiple electrode aggregometry (MEA, Multiplate, Roche diagnostics, Germany) with adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) as stimulants (Roche, Germany). Aggregation was measured as area under the Curve (AUC). In addition, thromboelastometry (ROTEM sigma, Werfen, Germany) was used to evaluate specific plasmatic coagulation parameters like FIBTEM A10, EXTEMCT and INTEM CT. As standard coagulation parameters, Quick, international normalized ratio (INR), and partial thromboplastin time (PTT) were determined.

Amount of erythrocyte transfusions.
To estimate the risk of perioperative bleeding the amount of erythrocyte transfusions was evaluated for each patient. The documented number of administered erythrocyte concentrates was counted from operating theatre and intensive care unit transfusion protocols.

Standardized bleeding assessment with thrombolysis in myocardial infarction (TIMI) bleeding criteria.
Bleeding was standardized with TIMI bleeding criteria. As criteria fatal bleeding, perioperative intercranial bleeding, reoperation after closure of sternotomy to control bleeding, transfusion of more than 5 packed red blood cells within 48 h and chest tube output more than 2 L within 24 h were evaluated.
One-year follow-up of clinical endpoints. Follow-up was conducted for clinical endpoints at 1 year after surgery. Major adverse cardiovascular and cerebrovascular events (MACCE) were assessed including stroke, death, myocardial infarction and bleeding events.       Fig. 1).

Discussion
The major findings of this study were that (A) ADP stimulated platelet aggregation is higher in SA+ compared to SA− IE patient. This was independently from age, gender and CRP. (B) This was abolished in patients with ASA medication. (C) ASA did not lead to an increased rate of bleeding and need for blood transfusion. SA has different virulence factors, which interact with coagulation factors and mediate platelet activation 8,10 . In human platelets, ClfA is considered as a main factor for activation of human platelets by SA 14 . ClfA is a staphylococcal surface protein that binds the C-terminus of human fibrinogens γ-chain 7,15 . The adherent bivalent www.nature.com/scientificreports/ fibrinogen molecule can bind to the platelets GPIIb/IIIa receptor and induce crosslinking 7,8 . As this crosslinking alone is insufficient to trigger platelet aggregation, a second stimulating agent is required 7 . Rapid platelet activation has been described for simultaneous binding of ClfA specific antibodies, linking ClfA to the platelets FcγRIIa-receptor, enhancing GPIIb/IIIa receptor signalling 7,16 . However, other mechanisms involving ClfA are conceivable. ADP is a main physiological agent to induce platelet aggregation through the P2Y12-and P2Y1-receptors, located on human platelets 17 . The P2Y12-receptor is known to activate GPIIb/IIIa receptors through a phosphoinositide-3-kinase (PI3-K) pathway 17 . While ClfA bound fibrinogen is presented to the GPIIb/IIIa receptor, its activation through ADP could have synergistic effects. This mechanism could lead to amplified platelet reactivity. Additionally, we conducted in-vitro experiments which showed that ADP mediated platelet aggregation can be amplified in presence of ClfA in healthy individuals ( Supplementary Fig. 2).
Besides ADP, thrombin is a strong mediator of platelet aggregation. Thrombin mainly activates human platelets through the PAR-1 receptor 18 . SA and other bacteria can induce endogenous thrombin formation through systemic inflammatory reactions 19,20 . This increase of endogenous thrombin can lead to a decrease of PAR-1 expression on platelets already at very low persistent concentrations 21 . The decrease of PAR-1 is described as time and dose dependent and could be shown in patients with systemic inflammation 21 . Ex-vivo, PAR1 downregulation comes with a decreased responsiveness to TRAP in MEA 21 . These two mechanisms could explain our findings that platelets of patients with coagulase positive IE had an increased responsiveness to ADP dependent platelet aggregation while TRAP dependent platelet aggregation did not differ in our study.
Beside of pathologically increased platelet activation by SA, other mechanisms should be considered. There is evidence that bloodstream infections with SA can lead to a massive inflammatory response 22 . In this context CRP, as common inflammatory marker, is increased. Studies showed that elevated CRP levels can affect platelet function 23,24 . Other factors that might influence human platelet aggregation and must be considered as confounding parameters are age and gender 25 . In our study, we identified that patients with SA+ IE were slightly younger and less often male compared to SA− patients. No other differences were detected between the study groups. But even after adjustments for age, gender and CRP our findings remained robust in multivariate analysis.
We also investigated thromboelastometry and coagulation parameters in patients with SA+ IE compared to patients with SA− IE. No significant differences could be shown, although previous studies suggest that SA impairs plasma clotting times and increases PTT by secreting extracellular soluble virulence factors Staphopain A and Staphopain B 26 .
Clinical studies have shown that patients with IE caused by SA show higher morbidity and mortality 2,27 . This primarily results from association of SA caused IE with higher rates of thromboembolic events leading to major neurological events and poor outcomes 28,29 . In this study we found that ASA treatment prior to surgery was associated with reduced 1 year mortality. However, this must be interpreted with caution. All patients had oral anticoagulation for 3 months after surgery. Additionally, the study size was not powered for multivariate analysis regarding clinical endpoints. Therefore, this has to be tested in randomized trials. However, it is well known that thromboembolic events have impact on prognosis of patients with SA+ IE, therefore antithrombotic medication might play a significant role to improve outcome 29 .
Previous research on IE could show that continuous ASA medication reduces the number of thromboembolic events and reduces vegetation size, confirming the importance of platelet reactivity [30][31][32][33] . However, data on this www.nature.com/scientificreports/ topic was controversial because some studies identified higher bleeding rates in IE patients on ASA therapy 34,35 . However, data did not show that ASA prevented thromboembolic events, which goes in line with our findings. Hence, the European Guidelines for treatment of IE do not yet recommend antiplatelet medication 36 . These studies did not investigate SA+ versus SA− IE patients. However, based on our results, patients with SA+ IE could particularly benefit of ASA medication. This is even underlined by the fact that ASA medication seemed to be safe in our study in SA+ patients. However, SA− patients with ASA had enhanced perioperative bleeding according to RBC transfusions. Postoperative bleeding, leading to reoperation also occurred more often in these patients. Although these findings were barely significant and case number was limited, it might underline the procoagulant effects of SA on the one hand and on the other hand emphasizes the safety of ASA medication in these patients.

Study limitations.
Our study has several limitations. First it was an observatory, single-center cohort study so external validity might be limited. There is a selection bias because only patients prior to surgical treatment were included, which might have led to a lower amount of SA+ patients. Indication for surgical treatment were not recorded. This could have influenced the results as valvular dysfunctions can impact platelet function tests. Furthermore, this study used point of care tests for evaluation of coagulation and platelet function that were available in our clinical setting. Therefore, we decided to carry out platelet function tests from citrated blood to meet the standards of our local laboratory, although hirudinized blood might be the standard for multiple electrode aggregometry. This might have influenced our findings. Also, it might have been interesting to further evaluate P-selectin levels and GPIIbIIIa activation which might have been more precise to evaluate platelet activation. However, these methods were not available in our daily clinical routine. Additionally, most of the patients did not reach the reference range of ADP induced platelet function test. Therefore, it is difficult to say if SA does increase platelet aggregation in general. However, we found that SA+ patients had higher platelet aggregation as compared to control. The trial was not randomized or controlled. Therefore, other potential confounders that were not included in the multivariable analysis might have biased the results.

Conclusion
This study showed that platelet aggregation is higher in ASA medication naïve patients with SA+ IE compared to patients with SA− E. This finding was abolished in patients with ASA medication, whereas bleeding was not enhanced in these patients. This leads to the hypothesis that ASA might be beneficial in SA+ endocarditis. Randomized clinical trials are needed to test this hypothesis.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.